MOLECULAR AND MORPHOLOGICAL CHARACTERIZATION OF SARCOCYSTIS INFECTIONS IN THE MUSCLES OF GRAY WOLVES (CANIS LUPUS) FROM MINNESOTA SUGGEST THEY MAY SERVE AS RESERVOIRS FOR INFECTION IN DOMESTICATED DOGS

Wolves are protected in the United States, except in Montana and Wyoming. Muscle samples were collected from 3 wolves in Minnesota under permit 35816 for specimen possession and research issued by the State of Minnesota Department of Natural Resources, Division of Fish and Wildlife, St. Paul, Minnesota. Details of wolves are summarized in Table II. The cause of death was unknown for the 2 wolves that were found dead. The third wolf was illegally shot but the perpetrator was not found.

Samples of the tongue and limb muscle were collected, put in Ziplock bags, and transported to the Animal Parasitic Diseases Laboratory (APDL), United States Department of Agriculture (USDA), Beltsville, Maryland for testing. Up to 9 days elapsed between collection and transport of samples to APDL (Table II).

Samples of muscles were fixed in 10% buffered formalin, embedded in paraffin, sectioned at 5 μm, stained with hematoxylin and eosin (HE), and examined microscopically for parasites. Parasites were photographed using an Olympus AX-70 microscope with a DP-73 digital camera and measurements were made digitally using Olympus Imaging Software cellSens Standard 1.18 (Olympus Optical Ltd., Tokyo, Japan).

Sarcocysts were identified in muscle squash preparation between a cover slip and a glass slide. The cover slip was removed, and the infected muscle piece was washed with McDowells Trump fixative (10% formalin 37%, 1% glutaraldehyde in 1.16% sodium phosphate monobasic and 0.27% NaOH with deionized water), and transported by air to Dr. Rafael Calero-Bernal, Complutense, University of Madrid, Spain for TEM examination; samples were postfixed as suggested by Kirillova et al. (2018). Ultrathin sections were examined at the Spanish National Centre for Electron Microscopy (Madrid, Spain) using a JEOL JEM 1400 Plus device at 80 kW.

The individual sarcocysts isolated from the 3 wolves were fixed in 95% alcohol and stored at −20 C until further use. Genomic DNA was extracted using the Qiagen DNeasy® Blood and Tissue Kit (Qiagen, Hilden, Germany) according to the instructions specified by the manufacturer and stored at −20 C. The DNA purity and concentration were evaluated by spectrophotometric analysis using Nanodrop (ThermoFisher Scientific, Waltham, Massachusetts). PCR amplification of highly conserved regions of 18S ribosomal RNA (18S rRNA), 28S ribosomal RNA (28S rRNA), a mitochondrial cytochrome c oxidase subunit 1 (COI), highly variable internal transcribed spacer-1 (ITS1) along with the rpoB gene (encoding the RNA polymerase β subunit) were performed using custom primers designed to amplify S. caninum and S. svanai (Table III). The β′ subunit is the largest subunit of RNA polymerase and is encoded by rpoC. PCR reactions totaling 25 μl consisted of 2 μl DNA template, 12.5 μl of Platinum Hot Start PCR Master mix (Invitrogen, Waltham, Massachusetts), 1 μl of 10 pmol/μl of each primer (Integrated DNA Technologies, Coralville, Iowa) (Table III) and 8.5 μl of molecular-grade water. After initial denaturation at 94 C for 3 min; 35 cycles were performed consisting of denaturation at 94 C for 30 sec, annealing at 60 C for 30 sec, and elongation at 68 C for 20 min; terminal elongation incubated products at 68 C for 5 min. The PCR products were analyzed on a 2% agarose gel and size was estimated by comparison with the 100–base-pair (bp) Plus DNA Ladder (Promega Corporation, Madison, Wisconsin). The PCR products were purified using the ExoSAP method (Bell, 2008). The final purified PCR products were subjected to bi-directional Sanger sequencing to Psomagen company (Rockville, Maryland) on an ABI 3500xl Genetic Analyzer (Applied Biosystems™, Waltham, Massachusetts) using the primer sets specially designed for this study.

Sequences were visualized, assembled, and edited using Geneious 11.1.5, and were submitted to GenBank. We compared sequences using the Basic Local Alignment Search Tool (BLAST) (Altschul et al., 1990). Each sequence showed 100% identity to either S. caninum or S. svanai (Table III).

Phylogenetic analyses were performed, independently, on nucleotide sequences of 18S rRNA, 28S rRNA, COI, ITS1, and rpoB gene. Each analysis included sequences obtained in this study as well as all sequences in the database with >95% homogeneity or above in the NCBI GenBank database using BLAST. The sequences identical to either S. caninum or S. svanai were also similar (but not identical) to each other: 99.42% (for 18S); 98.36% (for 28S); 99.16% (for COI); and 99.73% (for rpoB). We succeeded in amplifying the ITS1 only for S. svanai, which proved 99.30% identical to a reference sequence for S. caninum. We constructed multiple sequence alignments using Clustal W 2.1 (Larkin et al., 2007) and MAFT with default parameters as implemented in Geneious Prime® 2024.0.5. All sequences were trimmed at each end before phylogenetic reconstruction. To determine the most suitable nucleotide substitution, a model test was performed using jModelTest 2.1.7 (Darriba et al., 2012). Maximum-likelihood phylogenetic trees were constructed using MrBayes One Model (Ronquist et al., 2012) in TOPALi v2 (Milne et al., 2009) software having 1,000 bootstrap values. A total of 100,000 generations were taken for the phylogenetic tree. Included codon positions were 1st + 2nd + 3rd + Noncoding. All positions containing gaps and missing data were eliminated.

Sarcocysts were found in the muscles of all 3 wolves. Types 1 and 2 of sarcocysts were recognized based on the appearance of the sarcocyst wall (Fig. 1). Type 1 sarcocysts found in wolf 1 had a relatively thinner (<0.5 μm) cyst wall without any serrations (Figs. 1, 2). Sarcocysts were rare; 45 histological sections of 1 × 1–cm limb muscle yielded only 2 sarcocysts and none were found in the tongue. The 2 sarcocysts in HE-stained sections were 104 × 70 μm and 368 × 115 μm with a thin cyst wall (Fig. 2A). In unstained muscle squashes 27 cysts were found; the largest cyst was 3,450 × 138 μm.

Type 2 sarcocysts, from wolves 2 and 3, had a relatively thicker (>1 μm) cyst wall with serrations visible under optimal illumination of unstained squash preparations (Fig. 1B). The cyst wall in 1 unidentified sarcocyst had prominent molar tooth-like projections (Fig. 2C). In total, 52 sarcocysts were visible in unstained squashes (44 from wolf 2, 8 from wolf 3). The largest sarcocyst in unstained muscle squashes was 5,106 × 73 μm. One inflammatory focus associated with a degenerating sarcocyst-like structure was found in the limb muscle of wolf 2.

Four sarcocysts (2 from wolf 1 and 2 from wolf 2) were examined ultrastructurally. Both sarcocysts from wolf 2 were identical to S. caninum from the domestic dog (Dubey et al., 2015). The sarcocyst wall had undulating, pleomorphic villar protrusions (vp) on the sarcocyst wall like type 9c of Dubey et al. (2016). The vp lacked microtubules, and the ground substance was smooth with few scattered granules (Fig. 3).

By TEM, sarcocysts from wolf 1 appeared identical to sarcocysts of S. svanai from the domestic dog (Dubey et al., 2015). The wall was undulating (Fig. 4), like type 1a of Dubey et al. (2016); this type of sarcocyst wall (e.g., Sarcocystis muris) is thin and has blebs on the wall with a small stalk. However, in S. svanai, the wall invaginates into the ground substance (Fig. 4A); this feature was not noted in the original description of S. svanai of the dog (Dubey et al., 2015).

The final data set included 19 taxa and 1,563 positions for 18S; 18 taxa and 776 positions for 28S; 17 taxa and 939 positions for COI; 18 taxa and 1,541 positions for ITS1, and 17 taxa and 1,279 positions for rpoB. Homologous sequences from isolates of Sarcocystis myodes infecting Myodes glareolus were taken as the outgroup. The root was placed between the most distant of these (selected as the outgroup) and the rest (Fig. 5). The phylogenetic trees reconstructed based on maximum likelihood only differ in the position of a few low-supported branches (Fig. 5). These trees agree with previous studies on Sarcocystis spp. from carnivores (Gjerde and Schulze, 2014; Dubey et al., 2015; Calero-Bernal et al., 2016; Pavlásek and Máca, 2017; Hagner et al., 2018; Kirillova et al., 2018; Máca, 2018; Ye et al., 2018; Juozaityè-Ngugu et al., 2024) (Table I).

The phylogenetic tree based on all the genetic markers utilized during the study precisely formed 2 clades in almost all the trees: 1 with all the carnivores as intermediate hosts, and the other with birds as intermediate hosts with strong bootstrap support and almost the same branching topology. Furthermore, the phylogenetic tree formed 2 sister clades subdividing species infecting carnivores from species infecting birds, and then basal to that, the clade including Sarcocystis rileyi and Sarcocystis wenzeli. The tree in Figure 5A and D represents a slight departure from this result by the different placement of certain branches, for example, S. lari (accession number MF596283) and S. canis (accession number DQ176645). Also, the placement of the 2 major clades showed some minor variations depending on the sequence variability and alignment settings, or variations in the representative sequences. These variations influenced the positioning of the clade containing S. lari and S. canis to be placed in between subsets of species with carnivores and birds as intermediate hosts. Interestingly, S. caninum (present study) formed sister clades with Sarcocystis arctica, Sarcocystis canis, and Sarcocystis felis with high bootstrap values (Tables IV, V), because these species are closely related.

The phylogenetic evaluation confirmed that S. arctica cannot be genetically differentiated from S. caninum in the 4/5 loci studied. The phylogenetic analysis further showed that S. arctica and S. caninum were placed together with Sarcocystis species using carnivores as their intermediate host (S. canis, S. felis, and S. svanai) and establish the monophyletic relationship between them. However, as stated earlier they have different intermediate hosts (Table I). The phylogenetic also tree reiterates the importance of host specificity in these highly enigmatic parasites.

For reasons stated in the introduction, we wish to restrict the present discussion to Sarcocystis infections in the wolf. Sarcocystis infection was first described in an Alaskan wolf from the United States (Calero-Bernal et al., 2016). The Alaskan wolf (Canis lupus arctos) is a subspecies of the gray wolf, and its habitat is more geographically distinct than the gray wolf in the continental United States. Calero-Bernal et al. (2016) called the parasite S. arctica and did not discuss S. caninum. In retrospect, the parasite resembles S. caninum.

Here we provide the first record/description of Sarcocystis infections in the muscles of the gray wolf in Minnesota. Sarcocysts were found in all 3 wolves sampled. Although tissues were autolyzed, the structure of sarcocysts was preserved to obtain an ultrastructural description of the sarcocyst walls. The Sarcocystis species found were morphologically identical to S. caninum and S. svanai of the domestic dog (Dubey et al., 2015). Sarcocystis caninum and S. svanai have been associated with severe neuromuscular disorders in 2 dogs from British Columbia, Canada, 1 dog from Montana, and 1 dog from Colorado in the United States (Dubey et al., 2015), and 1 dog from Helsinki, Finland (Hagner et al., 2018). Of interest is that in 2 of the 5 dogs, S. caninum and S. svanai occurred concurrently, suggesting common epidemiological risk factors. Nothing was known concerning the cause of death in 2 wolves in the present study (Table I).

In the present study, only 1 species of Sarcocystis (either S. caninum or S. svanai) was found in each wolf. Domestic dogs and gray wolves are phylogenetically related and share common parasites (Lesniak et al., 2017). The occurrence of S. caninum and S. svanai from distant locations suggests that dogs and wolves are not accidentally infected with these Sarcocystis species but likely have an unrecognized cycle. There are not many predators of wolves to have a regular Sarcocystis cycle, but a wide range of scavenger species consume dead wolves or prey on young wolves and might act as definitive hosts.

Originally, S. svanai was discovered accidentally while reviewing TEM images of 2 dogs infected with S. caninum; therefore, a light-microscope description of the sarcocyst was unavailable (Dubey et al., 2015). Here, a light microscopic description of S. svanai sarcocyst was added both in unstained muscle squashes as well as in HE-stained sections. Additionally, we added to the ultrastructural description of the sarcocyst wall; the parasitophorous vacuolar membrane invaginated in ground substance, and here we designated the wall type as type 1a-1 to the wall type classification of Dubey et al. (2016).

Sarcocystis caninum and S. svanai infections were recently reported in gray wolves from Lithuania (Juozaityé-Ngugu et al., 2024) (Table I). Sarcocysts were detected in 4 (26.7%) of 15 gray wolves and sarcocysts were numerous. In 2 wolves, thin-walled S. svanai–like sarcocysts were detected, and in the other 2 wolves, thin-walled sarcocysts were mixed with remnants of S. caninum–like bradyzoites (Juozaityé-Ngugu et al., 2024); TEM was not performed. This investigation listed the pros and cons of different genetic markers for the diagnosis of S. caninum– and S. svanai–related Sarcocystis species (Table I).

Sarcocystis caninum and S. svanai were previously characterized molecularly, using loci such as 18S, 28S, COI, ITS1, and rpoB (Dubey et al., 2015). The phylogenetic analyses based on these loci provided insights into the relationships among Sarcocystis spp. infecting carnivores (intermediate hosts), and how they are related to birds and rodents highlighting the close genetic relationships between them, as well as their distinctness from other related species, shedding light on the host–parasite interactions and potential transmission patterns. In another report, S. caninum and S. svanai were molecularly characterized and described from a clinically affected dog (Hagner et al., 2018) in Finland; Pampas fox (Scioscia et al., 2017); 2 domestic dogs from China (Ye et al., 2018) and gray wolves from Lithuania (Juozaitytè-Ngugu et al., 2024). These studies show the importance of canids as a potential source of Sarcocystis spp. infections and their affinities to phylogenetically related host species in close proximity. Our data further implicate natural cycles, between wild canids and some (still unknown, possibly avian) definitive hosts, as reservoirs for infection in domesticated dogs.

The authors thank Dr. Petras Prakas for helpful suggestions, and Marisa García for excellent technical assistance on TEM procedures at the National Centre for Electron Microscopy, Madrid, Spain. This research was supported in part by an appointment of Aditya Gupta and Larissa Araujo to the Agricultural Research Service (ARS) Research Participation Program administered by the Oak Ridge Institute for Science and Education (ORISE) through an interagency agreement between the U.S. Department of Energy (DOE) and the U.S. Department of Agriculture (USDA). ORISE is managed by Oak Ridge Associated Universities (ORAU) under DOE contract DE-SC 0014664.